Application of EGCG (epigallocatechin gallate) in preparation of medicine for causing tumor immunogen cell death

Abstract

The invention discloses an application of EGCG (epigallocatechin gallate) in preparation of a medicine for causing tumor immunogen cell death. The invention discovers that the micromolecule EGCG causes immunogenic death of colon cancer cells for the first time; eGCG is combined with an immune checkpoint inhibitor in vivo for treatment, so that the obvious inhibition effect on occurrence and development of colon cancer is achieved; meanwhile, EGCG can induce death of immunogenic cells of colon cancer cells, so that the colon cancer whole-cell vaccine is prepared, and after mice are immunized with the colon cancer whole-cell vaccine, the survival rate of the colon cancer tumor-bearing mice can be effectively increased. EGCG improves the effect function of CD8T cells by increasing the maturation and infiltration of DC cells, enhances the anti-tumor curative effect of an immune checkpoint inhibitor, and provides a new method and a new thought for tumor treatment.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and medicine, and specifically relates to the application of EGCG in preparing a medicine for inducing tumor immunogenic cell death. Background Art

[0002] In recent years, tumor immunotherapy has made great progress, and immune checkpoint inhibitors have been shown to play an important role in a variety of tumors. However, this method still has many limitations, including the infiltration of tumor-infiltrating lymphocytes (TIL), the expression of PD-L1, high mutation and the expression of new antigens. Immunogenic antigens can be recognized by TIL, so enhancing the immunogenicity of tumors plays an important role in the effect of immunotherapy.

[0003] Immunogenic cell death (ICD) is a tumor cell death mode that can activate the body's anti-tumor response. Tumor cells undergoing immunogenic cell death can express a variety of related molecules, including Calreticulin (CRT), heat-shock protein 70 (HSP70) and High mobility group box 1 (HMGB1). In this process, CRT migrates from the endoplasmic reticulum to the cell membrane surface to attract phagocytes. In the middle stage, HSP70 transfers from the endoplasmic reticulum to the cell membrane surface. In the late stage, HMGB1 activates antigen presenting cells.

[0004] Tumor cells undergoing immunogenic cell death show decreased metabolic levels, endoplasmic reticulum stress, and apoptosis, thereby inhibiting tumor cell proliferation. Therapies targeting immunogenic cell death of tumor cells can effectively increase the responsiveness of T cells to tumors and enhance the efficacy of immune checkpoint inhibitors. Summary of the invention

[0005] The present invention discloses the use of EGCG in the preparation of a drug for inducing tumor immunogenic cell death, and aims to provide the use of epigallocatechin gallate EGCG in inducing tumor immunogenic cell death, enhancing T cell effector function, and enhancing the anti-tumor efficacy of immune checkpoint inhibitors. Through this study, a variety of new uses of EGCG were discovered, including inducing colon cancer immunogenic cell death, improving T cell reactivity, and the efficacy of immune checkpoint inhibitors in treating liver cancer. By inducing colon cancer immunogenic cell death, an anti-colon cancer whole cell vaccine was prepared, thereby improving the survival rate of colon cancer-bearing mice.

[0006] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0007] The invention discloses application of epigallocatechin gallate EGCG in preparing a drug for inducing tumor immunogenic cell death.

[0008] The invention discloses application of epigallocatechin gallate EGCG in preparing medicine for treating colon cancer.

[0009] As a further improvement, the medicine of the present invention is any one of an injection, an oral agent, a cream, and a spray.

[0010] The invention discloses the application of epigallocatechin gallate EGCG combined with CTLA-4 antibody in preparing a medicine for treating colon cancer.

[0011] The invention discloses application of epigallocatechin gallate EGCG in preparing a drug for increasing maturation and infiltration of DC cells and improving effector function of CD8 T cells.

[0012] The invention discloses application of epigallocatechin gallate EGCG in preparing whole-cell intestinal cancer vaccine for treating colon cancer.

[0013] The beneficial effects of the present invention are as follows:

[0014] 1) The present invention first discovered that EGCG can cause immunogenic cell death of colon cancer cells in vitro; EGCG can inhibit the growth of colon cancer cells by regulating gene expression, promoting biological processes such as endoplasmic reticulum stress, cell metabolism, cell apoptosis, cytokine secretion, and T cell immune response. This discovery provides a new target and mechanism for immunotherapy, which helps to overcome problems such as immune escape and immune tolerance;

[0015] 2) EGCG induces tumor immunogenic cell death, increases the maturation and infiltration of DC cells, and improves the effector function of CD8 T cells; that is, by enhancing the infiltration and maturation of DC cells and the effector function of CD8 T cells, the effect of immunotherapy is improved. This approach is expected to overcome the problems of existing immunotherapy that require high costs and complex operations.

[0016] 3) EGCG combined with immune checkpoint inhibitor CTLA-4 antibody improves tumor treatment effect. This discovery provides a new combination for immunotherapy, that is, by combining EGCG and CTLA-4 antibody to improve the effect of immunotherapy. Since EGCG and CTLA-4 antibody are both known drugs with good safety, this technical solution provides an immunotherapy with good safety.

[0017] 4) EGCG acts on mouse colon cancer cells and can be used to prepare whole-cell colon cancer vaccines. Immunization of mice can effectively improve the survival rate of mice, providing new methods and new ideas for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the use of EGCG to induce apoptosis of colon cancer cells in Example 1;

[0019] Figure 2 This is a diagram of immunogenic cell death induced by EGCG in colon cancer cells in Example 2;

[0020] Figure 3 This is a diagram showing the use of EGCG in preparing a whole-cell colon cancer vaccine to improve the survival rate of colon cancer-bearing mice in Example 3;

[0021] Figure 4 This is a diagram showing the use of EGCG in Example 4 to increase the maturation and infiltration of DC cells and improve the effector function of CD8 T cells;

[0022] Figure 5 This is a diagram showing how EGCG combined with immune checkpoint inhibitors improve the tumor treatment effect in Example 5. DETAILED DESCRIPTION

[0023] The invention discloses application of EGCG in preparing a drug for inducing tumor immunogenic cell death.

[0024] The invention discloses application of epigallocatechin gallate EGCG in preparing a medicine for treating colon cancer. The medicine is any one of an injection, an oral agent, a cream and a spray.

[0025] The invention discloses the application of epigallocatechin gallate EGCG combined with CTLA-4 antibody in preparing a medicine for treating colon cancer.

[0026] The invention discloses application of epigallocatechin gallate EGCG in preparing a drug for increasing maturation and infiltration of DC cells and improving effector function of CD8 T cells.

[0027] The invention discloses application of epigallocatechin gallate EGCG in preparing whole-cell intestinal cancer vaccine for treating colon cancer.

[0028] In order to better understand the present invention, the content of the present invention is further explained through specific embodiments in combination with the accompanying drawings below, but the content of the present invention is not limited to the following embodiments.

[0029] Example 1 EGCG was used to inhibit the activity of colon cancer cells and induce apoptosis.

[0030] The apoptosis-promoting effect of EGCG at different concentrations on a variety of colon cancer cells and the inhibition of their cell activity were determined by detecting cell apoptosis markers, including the following steps:

[0031] (1) Take cells in the logarithmic growth phase, digest them and count them using a hemocytometer;

[0032] (2) Add 5×10 3 cells, 100 μL per well; after 2-4 hours of attachment, different concentrations of EGCG were added for 24 hours;

[0033] (3) digesting the cells with trypsin and washing them, and incubating the cells with the corresponding antibodies;

[0034] (4) Flow cytometry was used to detect the expression of early and late apoptosis markers and calculate the proportion of cell apoptosis.

[0035] The inhibitory effect of EGCG on tumor cell proliferation obtained by the above method is as follows Figure 1 The results showed that EGCG treatment of various colon cancer cells in vitro could increase the expression of apoptotic factors on the surface of tumor cells and promote apoptosis of tumor cells.

[0036] Example 2: EGCG was used to inhibit the immunogenic cell death of colon cancer cells.

[0037] Various colon cancer cells were treated with different concentrations of EGCG, and the expressions of CRT and HSP70, as well as the secretion of HMGB1 were detected.

[0038] HMGB1 level detection:

[0039] 2×10 5 Tumor cells were seeded in each well of a 24-well plate and treated with EGCG for 24 hours. The cell culture medium was collected and the HMGB1 content in the culture medium was detected using an ELISA detection kit.

[0040] CRT and HSP70 detection:

[0041] 2×10 5 Tumor cells were seeded in each well of a 24-well plate and treated with EGCG for 24 hours. The cell culture medium was collected, the cells were washed, and then incubated with the corresponding antibodies, and the surface molecule expression was detected by flow cytometry.

[0042] The experimental results obtained according to the above method are as follows Figure 2 As shown, EGCG treatment of various colon cancer cells in vitro had an effect on tumor cell CRT ( Figure 2 AD), HSP70( Figure 2 EH), HMGB1( Figure 2The results showed that after EGCG treatment of tumor cells, the expression of CRT and HSP70 on the cell surface increased, and the amount of HMGB1 secreted by the cells into the external environment increased.

[0043] Example 3: EGCG is used to inhibit endoplasmic reticulum stress in colon cancer cells.

[0044] RNA-seq was performed on the drug-treated group and the control group, and differential gene analysis and signal pathway cluster analysis were performed.

[0045] The expression differences and clustering results obtained according to the above method are as follows Figure 3 A and Figure 3 As shown in B. Figure 4 As shown in A, EGCG-treated HT29 and SW480 colon cancer cell lines showed gene expression changes with some overlap ( Figure 3 A) Cluster analysis was performed based on differentially expressed genes. The results are shown in Figure 2. Figure 3 As shown in B: In the drug-treated group, endoplasmic reticulum stress, cell metabolism, apoptosis, cytokine secretion, and T cell immune response were highly enriched. The expression of endoplasmic reticulum stress-related genes increased significantly after EGCG treatment ( Figure 3 C), using RT-PCR to verify specific gene expression differences ( Figure 3 D).

[0046] Example 4 EGCG was used to promote the infiltration and maturation of DC cells and enhance the effector function of CD8 T cells.

[0047] (1) Establish a mouse immunotherapy model. The control group and the drug treatment group were inoculated with 5×10 5 CT26 tumor cell line, the drug group was injected with EGCG, and the control group was injected with PBS to construct the tumor model;

[0048] (2) After 12 days, the mice were killed, and the tumor tissues were obtained by enzymatic hydrolysis to obtain single cells;

[0049] (3) Flow cytometry was used to analyze the infiltration of T cells (changes in the proportion of CD8 and CD4 positive cells) and DC cells (changes in the proportion of CD11c positive cells), T cell effector function (changes in the expression of IFN-γ and TNF-α), and DC cell maturation (changes in the expression of CD80, CD86, and MHC-II);

[0050] The experimental results obtained according to the above method are as follows Figure 4 As shown, in the EGCG-treated group, DC cell infiltration increased ( Figure 4 B), increased DC cell maturation ( Figure 4 D), T cell infiltration did not change significantly ( Figure 4C), but the effector function of CD8 T cells is increased ( Figure 4 E).

[0051] Example 5

[0052] Using EGCG combined with immune checkpoint inhibitor CTLA-4 antibody to improve tumor treatment effect:

[0053] 1. EGCG combined with immune checkpoint inhibitor CTLA-4 antibody to improve the body's immunotherapy effect on tumors

[0054] (1) Establish a mouse immunotherapy model. The control group and the drug treatment group were inoculated with 5×10 5 CT26 tumor cell line, used to construct tumor model;

[0055] (2) One week later, EGCG: 30 mg / kg, once a day, for 12 consecutive days; immune checkpoint inhibitor CTLA-4 antibody: 200 μg / time, once every 3 days, for a total of 3 times;

[0056] (3) Observe the changes in tumor volume every day;

[0057] The experimental results obtained according to the above method are as follows Figure 5 As shown, in immunotherapy experiments ( Figure 5 A), compared with the control group, EGCG combined with CTLA-4 antibody treatment of mice can effectively inhibit the growth of tumors in vivo ( Figure 5 B), providing a mechanistic basis for combined therapy.

[0058] Obviously, the above embodiments are merely examples for clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from this are still within the protection scope of the invention.

Claims

1. Application of EGCG in the preparation of drugs for inducing tumor immunogenic cell death.

2. Application of EGCG in the preparation of drugs for the treatment of colorectal cancer.

3. The use according to claim 2, characterized in that: The medicine is any one of an injection, an oral agent, a cream, and a spray.

4. Application of EGCG combined with CTLA-4 antibody in the preparation of drugs for the treatment of colorectal cancer.

5. Application of EGCG in the preparation of drugs for increasing the maturation and infiltration of DC cells and improving the effector function of CD8 T cells.

6. Application of EGCG in the preparation of whole-cell colorectal cancer vaccine for the treatment of colon cancer.

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